Lymphocyte trafficking is governed in part by a host of chemotactic signals encountered in lymphoid organs and peripheral tissues. How the quantitative characteristics of chemokine gradients derived from 'point'sources (the physiologic setting of attractants secreted from discrete cells) control the 3D navigation of lymphocytes through tissues and their localization in secondary lymphoid organs (SLO) remains poorly defined. We propose to use a simplified in vitro model system to quantitatively examine how lymphocytes interpret chemokine gradients released from local sources. Synthetic hydrogel microspheres that encapsulate and slowly release chemokines at predetermined rates will be used to engineer chemokine gradients in collagen gel surrogate ECMs. By fluorescently labeling human T and B lymphocytes and encapsulating a fluorescently-labeled 'tracer1 quantity of chemokine in microspheres, we will simultaneously track by videomicroscopy lymphocyte migration and the evolution of chemokine gradients over time. We will use this system to dissect aspects of T cell and B cell chemotaxis relevant to homeostasis and antigen priming in the SLO: (1) We will determine how lymphocyte migration is modulated by isolated chemokine point sources as a function of chemokine gradient characteristics. (2) To understand how lymphocytes respond to clusters of spatially discrete chemokine sources, we will use chemokine-releasing microspheres dispersed at varying density within a defined volume of a collagen matrix to determine how lymphocyte navigation through 3D volumes is influenced by the presence of multiple point sources of chemokine. (3) Using physically adjacent depots of CXCL13- and CCL21-releasing microspheres, we will determine how T and B lymphocyte self-organization is controlled by chemokine gradient characteristics, and characterize how lymphocyte activation alters T and B cell trafficking at a CXCL13/CCL21 gradient interface as a function of time and gradient characteristics. These studies will reveal the quantitative requirements for soluble chemokine gradients to induce lymphocyte chemotaxis and control lymphocyte positioning, in a system modeling the physiologic setting of discrete, 'point'-like chemokine sources. These results will have implications for understanding how trafficking in the immune system is regulated and will shed light on how chemokines might be targeted to inhibit or enhance ectopic lymphoid tissue formation for immunotherapy.